We have previously found that 22:6n-3 promotes the accumulation of phosphatidylserine (PS) and prevents apoptotic neuronal cell death. During this period, the effect of polyunsaturates on the survival of neuronal cells was investigated along with the underlying mechanisms of this effect. We demonstrated that 22:6n-3 prevented the apoptotic cell death of both Neuro 2A and PC-12 cells, only after a prolonged period of enrichment, suggesting that the protective effect of 22:6n-3 may be exerted as a membrane phospholipid constituent. Under conditions where PS accumulation is inhibited, 22:6n-3 enrichment was not effective. In addition to PS accumulation, growth factor signaling pathways such as Raf-1 and PI3-kinase pathways appeared to be important for the protective effect of 22:6n-3. Translocation of Raf-1, an important upstream kinase transducing growth signaling, to membrane was significantly enhanced in 22:6n-3 enriched cells. According to in vitro biomolecular interaction analysis, the interaction between unilamellar vesicles of phospholipids and Raf-1 kinase required the presence of PS in the vesicle and the extent of interaction was indeed dependent on the PS composition. During n-3 fatty acid deficiency or chronic alcohol exposure, we found that the PS content can be markedely decreased specifically in neuronal cells where 22:6n-3 was highly enriched. These results support the view that PS accumulation promoted by 22:6n-3 plays an important role in growth factor signaling and deprivation of this fatty acid either due to n-3 fatty acid dietary deficiency or due to chronic alcoholism may have adverse effects on neuronal survival. We also found during this period that melatonin, the major product of the pineal gland, affects polyunsaturated fatty acid metabolism by down-regulating cytosolic PLA2 at gene and protein expression levels. These data suggest that polyunsaturated fatty acids may be involved in biochemical functions of melatonin.